Nutrient pharmaceutical formulation comprising polyphenols and use in treatment of cancer

ABSTRACT

A nutrient pharmaceutical formulation composition useful for treating cancer comprising ascorbic acid, L-lysine, L-proline and at least one polyphenol compound selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin, catechin and the use of the composition in treating cancer and other tumors are provided. A nutrient pharmaceutical formulation composition of EPICAN FORTE™ and its method of use in treating cancer are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application Ser. No. 60/348,143 filed Jan. 11, 2002, thecontent of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a nutrient pharmaceutical formulationand its use for the treatment of cancer. More specifically, the presentinvention relates to polyphenol containing pharmaceutical formulationshaving an effective amount of polyphenols for the treatment of cancer.The formulation of the present invention is used as an agent for theprevention and treatment of cancer comprising ascorbic acid, lysine,proline and at least one polyphenol compound selected from the groupconsisting of epigallocatechin gallate, epicatechin gallate,epigallocatechin, epicatechin, and catechin.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the industrializedworld. There exists no specific and causal treatment for cancer andmortality from cancer is among the highest from all diseases. The mostwidely used treatments—chemotherapy and radiotherapy—do not distinguishbetween healthy tissue and cancer and are associated with severe sideeffects. Thus, there is a need for a selective treatment of cancer.

One of the key mechanisms that cancer cells use in order to expand andmetastasize in the body involves enzymatic destruction of thesurrounding connective tissue. Therapeutic approaches to control thisprocess through specific drugs have not been successful and currentlythere are no means available to control cancer metastasis. Currenttreatment protocols with chemotherapy and radio-therapy focus on cancercell destruction in the body, and they do not address metastasis.Moreover, these treatments are toxic, not specific and associated withsevere side effects. Chemotherapy and radio-therapy carry a risk of thedevelopment of new cancers and through their destruction of connectivetissue in the body can facilitate the invasion of cancer.

In order to grow and expand to other parts of the body, cancer cellsdegrade the extracellular matrix through various matrixmetalloproteinases (MMPs) and plasmin, whose activities have beencorrelated with an aggressiveness of tumor growth. Rath and Pauling(1992) postulated that nutrients such as an amino acid, lysine andascorbic acid can act as natural inhibitors of extracellular matrixproteolysis and as such they have the potential to modulate tumor growthand expansion. These nutrients can exercise their anti-tumor potentialthrough several mechanisms, among them by inhibition of MMPs and bystrengthening of the connective tissue surrounding cancer cells (tumor“encapsulating” effect).

U.S. Pat. No. 5,962,517 discloses a pharmaceutical composition for adifferent medical indication (acne treatment). The disclosed compositioncomprises an acne reduction component, at least one of burdock rootyellow dock root, or a catechin-based composition; and a skin cellconditioning component comprising a transition metal. The disclosedcomposition is not shown to have any beneficial value in cancertreatment and/or prevention.

PCT WO 00/76492 discloses a nutrient formulation for disease reductionthat contains a catechin compound. However, the bioavailability ofcatechin compounds has been shown to be small low (Chen L., Lee M. J.,Yang C. S. Drug Metab. Dispos. 25: 1045-1050 (1997); Yang C. S., ChenL., Lee M. J., Balentine D. A., Kuo M. C., Schantz S. Cancer Epidemol.Biomark. Prev. 7: 351-35 (1998); Bell J. R., Donovan J. L., Wong R.,Waterhouse H., German J. B., Walzem R. L., Kasim K. Am. J. Clin. Nutr.71:103-108 (2000); Sherry Chow H. H., Cai Y., Alberts D. S., Hakim I.,Dorr R., Shahi F., Crowell J. A., Yang S. C., Hara H. Cancer Epidemol.Biomark. Prev. 10: 53-58 (2001)) and the PCT WO 00/76492 fails todisclose a means to increase the bioavailability as is required incancer treatment and/or prevention.

Demeule et al. discloses that green tea catechins may have inhibitoryeffects on matrix metalloproteinase. There is no suggestion or teachingregarding how to use catechins in cancer treatment and/or prevention.Given the fact that the bioavailability of polyphenols in humans isextremely low, the low tissue concentration of catechins greatly reducesthe therapeutic value of polyphenols including epigallocatechin gallate(EGCG). There is a constant need in finding a better nutrientpharmaceutical composition containing polyphenols that is effective inthe treatment of neoplastic diseases and other diseases includinginflammatory diseases.

There is a need for safe and effective natural approaches that can beused to control the process of cancer expansion in the body. There isalso a need for a preventive measure against cancers or benign tumors todevelop in human and such measure could be applied to patients withoutthe risk of treatment-related side effects. Nutrient pharmaceuticalcompositions have become popular, as the incidence of cancer in recenttime increases. The requirement of such composition will continue andwill likely increase.

SUMMARY OF THE INVENTION

In one object, the present invention provides a nutrient pharmaceuticalcomposition useful in treating cancer, comprising: a) an ascorbiccompound, b) a L-lysine compound, c) a L-proline compound, and d) atleast one polyphenol compound selected from the group consisting ofepigallocatechin gallate, epicatechin gallate, epigallocatechin,epicatechin, and catechin. Compounds of a-c) enhance the polyphenolcompound's activity in blocking cancer proliferation and metastasis.

Preferably, the ascorbic compound is selected from the group consistingof ascorbic acid, pharmaceutical acceptable ascorbate salts, ascorbateesters and/or mixture thereof. Preferably, the pharmaceutical acceptableascorbate salt is calcium ascorbate or magnesium ascorbate. Morepreferably, the ascorbate ester is ascorbyl palimitate. Preferably, thelysine compound is selected from the group consisting of lysinehydrochloride and lysine pharmaceutically acceptable lysine salts.Preferably, the proline compound is selected from the group consistingof proline hydrochloride and proline pharmaceutical acceptable prolinesalts.

In another object, the present nutrient pharmaceutical compositionfurther comprises a trace element selected from the group consisting ofselenium, copper, manganese, calcium and magnesium.

In another object, the present nutrient pharmaceutical compositionfurther comprises an amino acid. Preferably, the amino acid is arginine.More preferably, the present nutrient pharmaceutical composition furthercomprises N-acetyl cysteine.

In another object, the present invention provides a nutrientpharmaceutical composition useful in treating cancer, comprising: a) anascorbic compound, b) a L-lysine compound, c) a L-proline compound, d)N-acetyl cysteine, and e) at least one polyphenol compound selected fromthe group consisting of epigallocatechin gallate, epicatechin gallate,epigallocatechin, epicatechin, and catechin. The a-d) compounds enhancethe polyphenol compound's activity in blocking cancer proliferation andmetastasis.

In another object, the present invention provides a nutrientpharmaceutical composition comprising 250 mg ascorbic acid, 250 mgcalcium ascorbate, 250 mg magnesium ascorbate, 250 mg ascorbylpalmitate, 1,000 mg polyphenols, 200 mg N-acetyl-cysteine, 1,000 mglysine, 750 mg proline, 500 mg arginine, 30 mcg selenium, 2 mg copper,and 1 mg manganese.

In another object, the present invention provides a nutrientpharmaceutical composition comprising 25 mg ascorbic acid, 25 mg calciumascorbate, 25 mg magnesium ascorbate, 25 mg ascorbyl palmitate, 200 mgpolyphenols, 10 mg N-acetyl-cysteine, 50 mg lysine, 25 mg proline, 50 mgarginine, 1 mcg selenium, 20 mcg copper, and 50 mcg manganese.

In another object, the present invention provides a nutrientpharmaceutical composition comprising 5,000 mg ascorbic acid, 5,000 mgcalcium ascorbate, 5,000 mg magnesium ascorbate, 5,000 mg ascorbylpalmitate, 5,000 mg polyphenols, 1,500 mg N-acetyl-cysteine, 5,000 mglysine, 3,000 mg proline, 3,000 mg arginine, 200 mcg selenium, 9 mgcopper, and 10 mg manganese.

In another object, the present invention provides a nutrientpharmaceutical composition comprising 250 mg ascorbic acid, 250 mgcalcium ascorbate, 250 mg magnesium ascorbate, 250 mg ascorbylpalmitate, 1,000 mg polyphenols, 200 mg N-acetyl-cysteine, 1,000 mglysine, 750 mg proline, 500 mg arginine, 100 mcg selenium, 2 mg copper,1 mg manganese, 500 mg calcium, and 400 mg magnesium.

In another object, the present invention provides a nutrientpharmaceutical composition comprising 250 mg ascorbic acid, 250 mgcalcium ascorbate, 250 mg magnesium ascorbate, 250 mg ascorbylpalmitate, 1,000 mg polyphenols, 200 mg N-acetyl-cysteine, 1,000 mglysine, 750 mg proline, 500 mg arginine, 100 mcg selenium, 2 mg copper,1 mg manganese, 500 mg calcium, 400 magnesium, and 200 citrusbiofavonoids.

In yet another object, the present invention provides a nutrientpharmaceutical composition useful in treating cancer, comprising:L-lysine, L-proline, L-arginine, ascorbic acid, calcium, magnesium,polyphenols, N-acetyl-cysteine, selenium, copper, and manganese.

In another object, the present invention provides a nutrientpharmaceutical composition comprising 1,000 mg L-lysine, 750 mgL-proline, 500 mg L-arginine, 710 mg ascorbic acid, 22 mg calcium, 50 mgmagnesium, 1,000 mg polyphenols, 200 mg, N-acetyl cysteine, 30 mcgselenium, 2 mg copper, and 1 mg manganese.

In another object, the present invention provides a method of treatingcancer in an individual, comprising the step of administering to theindividual the nutrient pharmaceutical compositions as disclosed.Preferably, the cancer is selected from the group consisting of melanomacancer, breast cancer, colon cancer, lung cancer and brain cancer.

In another object, the present invention provides a method of treatingan inflammatory disease in an individual, comprising the step ofadministering to the individual the nutrient pharmaceutical compositionas disclosed. Preferably, the inflammatory disease is osteoarthritis.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the inhibitory effects of polyphenols, ascorbate, prolineand lysine on migration behavior of human breast cancer cells.

FIG. 2 depicts the inhibitory effects of polyphenols, ascorbate,proline, and lysine on migration behavior of human colon cancer cells.

FIG. 3 depicts the inhibitory effects of polyphenols, ascorbate,proline, and lysine on migration behavior of human melanoma cells.

FIG. 4 depicts the inhibitory effects of polyphenol, ascorbate, proline,and lysine on apoptosis of human melanoma cells.

FIG. 5 depicts the inhibitory effects of ascorbic acid, proline, lysineand various amounts of EGCG on cell proliferation of human melanomacells (A 2058).

FIG. 6 depicts the inhibitory effects of EGCG on cell proliferation ofhuman breast cancer cells (MDA-MB 231).

FIG. 7 depicts the effects of supplementation of basal medium withascorbic acid, proline, lysine with various amounts of EGCG on cellproliferation of human breast cancer cells (MDA-MB 231).

FIG. 8 depicts the effects of supplementation of basal medium withascorbic acid, proline, lysine and various amounts of EGCG on cellproliferation of human colon cancer cells (HCT 116).

FIG. 9 depicts the effects of supplementation of basal medium withascorbic acid, proline, lysine and various amounts of EGCG on expressionof matrix metalloproteinases (MMP) in human melanoma cells.

FIG. 10 depicts the effects of supplementation of basal medium withascorbic acid, proline, lysine and various amounts of EGCG on Matrigelinvasion by human breast cancer cells (MDA-MB 231).

FIG. 11 depicts the effects of supplementation of basal medium withascorbic acid, proline, lysine and various amounts of EGCG on Matrigelinvasion by human colon cancer cells (HTC 116).

FIG. 12 depicts the effects of supplementation of basal medium withascorbic acid, proline, lysine and various amounts of EGCG on reducingthe number of invading cells in human melanoma cells (A 2058).

FIG. 13 depicts MR scans of a female patient diagnosed with Gliablastomaafter suffering a facial stroke. The left diagram reveals a brain tumoron the left side of the brain. The right diagram reveals that the braintumor disappeared after the administration of EPICAN FORTE™.

FIG. 14 depicts X-ray CT scans of a male patient diagnosed with prostatecancer. The left diagram reveals metastasis along the lymphatic vesselsof the aorta. The right diagram reveals that the lymphatic metastasiswas not detectable and that the patient's prostate gland returned tonormal size after the administration of EPICAN FORTE™

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “lysine” is used interchangeable with L-lysine,“proline” is used interchangeable with L-proline, “arginine” is usedinterchangeable with “L-arginine”; “vitamin C” is used interchangeablewith ascorbic acid, and may include ascorbate salts or ascorbate esters.“MMPs” refers to matrix metalloproteinases; e.g., MMP-1, MMP-2, MMP-3,MMP-4, MMP-5, MMP-6, MMP7, MMP-8, MMP-8, MMP-9, MMP-10, MMP-11 etc.“EGCG” refers to (−)-epigallocatechin-3-gallate which is the majorpolyphenolic constitutents present in green tea. “NHDF” refers to normalhuman dermal fibroblast, including human chondrocytes and human stromalcells.

The present invention provides a nutrient pharmaceutical compositionascorbic acid, lysine, proline and at least one polyphenol compound.Preferably, ascorbate compound is selected from the group consisting ofascorbic acid, ascorbate salts and ascorbate esters. Preferably, lysineis lysine hydrochloride or pharmaceutically acceptable lysine salts.Preferably, proline is proline hydrochloride or pharmaceuticallyacceptable proline salts thereof.

Polyphenol compounds are extracts of green tea. They are also known ascatecins. Polyphenols are present in green tea and have been suggestedto provide protection against variety of illnesses including cancer(Mukhtar H., Ahmed N. Am. J. Clin. Nutr. 71:1698S-1702S (2000)). Oraladministration of green tea enhanced the tumor-inhibitory effects ofdoxorubicin in mice. The potential anti-cancer activity of catechins mayrelate to their effects on several factors involved in proliferation ofcancer cells and their metastasis. Catechins are known to cause cellcycle arrest in human carcinoma cells (Ahmad N., Feyes D. K., NieminenA. L., Agarwal R., Mukhtar H. J. Natl Cancer Inst. 89: 1881-1886(1997)). Polyphenolic fraction from green tea is also implicated toprotect against inflammation and cytokines induced by tumors.

Polyphenolic compounds present as 30% dry weight in green tea. Theyinclude flavanols, flavandiols, flavonoids, and phenolic acids.Flavanols are the most abundant among the polyphenols in green tea andare commonly known as catechins. There are four major catechins in greentea: 1) (−)-epicatechin, 2) (−)-epicatechin-3-gallate, 3)(−)-epigallocatechin, and 4) (−)-epigallocatechin-3-gallate (EGCG).Among the catechins, EGCG is the major polyphenolic constitutentspresent in green tea. EGCG is a potent anti-oxidant compound and mayattribute to the anti-cancer activity of green tea. Catechin compoundswere reported to exercise its anti-metastatic activity by preventing theangiogenesis process (Cao Y., Cao R. Nature 398:381 (1999)). EGCG hasalso been shown to interfere with the activity of urokinase(u-plasminogen activator) (Jankun J., Selman S. H., Swiercz R.,Skrzypczak J. E. Nature: 387-567 (1997)), one of the most frequentlyexpressed enzymes in human cancers. EGCG is a preferred polyphenolcompound.

Preferably, polyphenol compound is selected from the group consisting ofepigallocatechin gallate, epicatechin gallate, epigallocatechin,epicatechin, catechin and other pharmaceutically acceptable polyphenolsalts and/or mixtures thereof.

One of the main therapeutic target of this patent application is theprevention of the digestion of the extracellular matrix and itsrestoration. Ascorbic acid or its salt (i.e., ascorbate) is required forthe synthesis of collagen, elastin and other key extracellular matrixmolecules.

Preferably, a combination of ascorbic acid, proline and lysine is usedto enhance the anti-cancer activity of polyphenol compounds. Morepreferably, the combination enhances the polyphenol compounds includingepigallocatechin gallate, epicatechin gallate, epigallocatechin,epicatechin, catechin in such a manner that the polyphenol compound iseffective in reducing invasion of cancer cells or completely blockingthe invasion.

Without bound by any theory, the present nutrient pharmaceuticalformulation contains a mixture of ingredients including polyphenols andthe mixture is found to function effectively in blocking proliferationand metastasis processes. We found that the present nutrient formulationcomposition significantly reduces the invasion of cancer cells or blocksthe metastasis of cancer cells completely. Thus, this compositioneffectively prevented these cancer cells from spreading. The therapeuticuse of the compound claimed is an effective, selective and safetreatment for cancer of the breast, colon and of other organs.Preferably, the disclosed nutrient pharmaceutical composition maycontain two of the components that are covalently bound.

Preferably, the efficacy of the nutrient composition is enhanced byincluding compounds known to beneficially affect growth and invasion ofcancer and other tumors, including L-arginine and/or arginine-containingcompounds. More preferably, N-acetyl-cysteine is used. More preferably,a selenium salt, a copper salt, and a manganese salt are used that areeffective to prevent and treat cancer and other tumors. This compositionmay also include one or more compounds needed as a coenzyme in theKrebs-Cycle, the respiration chain, or for other metabolic functions ofcells in an amount effective to prevent and treat cancer and othertumors.

The present nutrient pharmaceutical composition may be administered to apatient in form of capsules, tablets, powders, pills, injections,infusions, inhalations, suppositories or other pharmaceuticallyacceptable carriers and/or means of delivery for the prevention andtreatment of cancer and other tumors. Preferably, the nutrientpharmaceutical composition is administered as capsules, tablets orpowders. More preferably, it is administered as capsules.

The present nutrient pharmaceutical composition can be used inpreventing and treating cancer and other tumors in a selected patientwhere the tumor or cancer is located in the breast, ovaries, cervix orother organs of the female reproductive system, lung, liver, skin,gastrointestinal system, brain, bones or other organs of the body.Preferably, the composition is used in lung cancer and brain cancer.

The present nutrient pharmaceutical composition can also be used inpreventing and treating the infectious diseases, atherosclerosis,restenosis, other cardiovascular diseases and inflammatory diseases.Preferably, the inflammatory disease is osteoarthritis.

MMP-Mediated Plasmin Activation: It is speculated that MMPs activity canbe affected by lysine through plasmin-mediated mechanisms although othermechanisms are not excluded. The MMPs are secreted as proenzymes andtheir activation is mediated partially by plasmin and its completionrequires active form of MMP-3. The mechanism of activation of variousMMPs detailed by Nagase (1997) indicates that MMP-3 also requires theconversion of plasminogen to its active form, plasmin. Plasminogenactive binding center has the sites where lysine gets specificallyattached. Therefore lysine can interfere with activation of plasminogeninto plasmin by plasminogen activator (Rath and Pauling, 1992), bybinding to plasminogen active sites. tranexamic acid, a synthetic lysineanalog has been used to inhibit plasmin induced proteolysis through thismechanism.

Since plasmin activity is essential to induce several tissue MMPs,lysine can interfere with conversion of plasminogen to plasmin andthereby it can inhibit the activation of almost all MMPs. In addition,EGCG may exert an inhibitory effect on extracellular matrix degradationthrough inhibition of MMP-2.

Cancer Invasion and Role of Matrix: It is also possible to affect cancercell matrix invasion by increasing the stability and strength of theconnective tissue surrounding cancer cells, and contributing to the“encapsulation” of the tumor. This requires optimizing the synthesis andstructure of collagen fibrils, for which the hydroxylation ofhydroxyproline and hydroxylysine residues in collagen fibers isessential. Ascorbic acid may be essential for hydroxylation of theseamino acids. Ascorbic acid and L-lysine are not normally produced in thehuman body; therefore, suboptimal level of these nutrients is possiblein various pathological stages as well as through inadequate diets.Although proline can be synthesized from arginine, its synthesis and orhydroxylation may be affected at pathological conditions. As such, ithas been shown that hydroxyproline content of metastatic tumor tissue ismuch lower than non-metastatic tumor tissue (Chubainskaia et al., 1989).A variety of drugs that reduced metastasis also increased thehydroxyproline content of the tissues (Chubinskaia et al., 1989). Theurinary hydroxyproline content of cancer patients has been found to behigher than that in healthy persons or non-cancer patients (Okazaki etal., 1992). All these findings suggest adverse effects of cancer cellson metabolism of proline and possible conditioned deficiency of prolinein cancer patients.

Cancer patients may have insufficient levels of ascorbic acid. Ascorbicacid may be cytotoxic to malignant cell lines and exerts anti-metastaticaction. The present invention discloses that a combination of ascorbicacid, proline, lysine and at least one poyphenol compound exerts apotent anti-proliferative and anti-metastatic effect on cancer celllines. Preferably, the present nutrient pharmaceutical formulation iseffective against melanoma, breast cancer and colon cancer cells. Mostpreferably, the present pharmaceutical formulation is effective againsthuman colon cancer cells.

Polyphenol Compounds (Catechins): EGCG represents one of the catechinsin green tea extract and may have growth inhibitory effects againsthuman cancer cells. The exact underlying mechanism is unclear. Thepresent invention discloses a surprising synergy in the nutrientpharmaceutical formulation comprising ascorbic acid, proline and lysineand at least one polyphenol compound in effectively blockingproliferation and metastasis of cancer cells.

The nutrient pharmaceutical composition of the present inventioneffectively block the invasion of cancer of the breast, colon, skin(melanoma), and other forms of cancer. The ingredients present in thenutrient formulation are naturally occurring compounds; and when used inthe claimed range as disclosed in the application, they are shown tohave no toxic side effects. Thus, this composition can also be usedprophylactically, i./e. the effective prevention of cancer and othertumors in the body.

Since viral cells and other invasive microorganisms use similarproteases that cancer cells for spreading of the infection through thebody, the composition claimed in this patent may be used in theeffective prevention and treatment of viral diseases and otherinfectious diseases.

Similar mechanism of activation of matrix metalloproteinases that cancercells use for matrix invasion also contributes to destabilization ofatherosclerotic plaques leading to myocardial infarctions and stroke.Therefore, the composition claimed in this patent can also be used inthe effective prevention and treatment of atherosclerosis, restenosisand other cardiovascular complications.

Activation of matrix metalloproteinases that cancer cells use for matrixinvasion is a key component in various inflammatory conditions.Therefore, the composition claimed in this patent can also be used inthe effective prevention and treatment of diseases, such as rheumatoidarthritis, emphysema, allergies, osteoarthritis and other conditionsthat include inflammatory aspects.

The nutrient pharmaceutical composition of the present invention can beprovided to a patient in form of tablets, pills, injections, infusions,inhalations, suppositories or other pharmaceutically acceptable carriersand/or means of delivery.

EXAMPLES

The effects of ascorbic acid, lysine, proline, and at least onepolyphenol compound were studied for their anti-proliferative andanti-invasive potential in various human cancer cell lines. Moreparticularly, one of the green tea extract ingredient (i.e.,epigallochatechin gallate (EGCG)) was examined.

Materials and Methods

Human breast cancer cells MDA-MB-231, human colon cancer cells HCT 116,human melanoma cell line A2058 were obtained from ATCC. Normal humandermal fibroblasts were obtained from GICBO. Where not indicated, theculture media obtained from ATCC were used.

In cancer cell proliferation studies each treatment was replicated eighttimes. In the invasion assays, each treatment was performed in three orfour replicates.

Cell Proliferation Studies

In these studies 5×10⁴ breast cancer cells were grown in Liebovitz'smedium with 10% fetal bovine serum (FBS) in 24-well plates. The mediumwas used as such (basal) or with designated supplements. Plates wereincubated in an air-circulating incubator (without supplemental CO₂) forfour days. The colon cancer cells HCT116 were grown in McCoy's 5A mediaand maintained in a 5% CO₂-air circulating incubator. At the end of theincubation period, the media were withdrawn and the cells in the wellswere washed with PBS followed by incubation for 3 hours with MTT stain.Dimethyl-sulfoxide (DMSO, 1 ml) was added to each well. The plates withDMSO were allowed to stand at room temperature for 15 minutes withgently agitation and then OD of the solution in each well was measuredat 550 nm. The OD₅₅₀ of the DMSO solution in the well was considered tobe directly proportional to the number of cells. The OD₅₅₀ of treatmentthat did not contain any supplement (Basal) was considered as 100.

Matrigel Invasion Studies

The studies were conducted using Matrigel (Becton Dickinson) inserts incompatible 24-well plates. Fibroblasts were seeded and grown in thewells of the plate using DMEM. When fibroblasts reached confluence, themedium was withdrawn and replaced with 750 μl of the media designatedfor the treatment. The cancer cells (5×10⁴) suspended in 250 μl of themedium supplemented with nutrients as specified in the design of theexperiment were seeded on the insert in the well. Thus both the mediumon the insert and in the well contained the same supplements. The plateswith the inserts were then incubated (in air-circulating incubator forMDA-MB-231 cells and 5% CO₂ incubator for colon cancer cells andmelanoma cells) for 18-20 hours. After incubation, the media from thewells was withdrawn. The cells on the upper surface of the inserts weregently scrubbed away with cotton swab. The cells that had penetrated theMatrigel membrane and had migrated on the lower surface of the Matrigelwere stained with hemacolor stain (EM Sciences) and were visuallycounted under the microscope. The results were subjected to ANOVA andall the possible pairs were tested for significance at p<0.05.

The media in different studies were supplemented with ascorbic acid,proline, lysine and EGCG at concentrations as indicated.

Gelatinase Zymography

Gelatinase zymography was performed in 10% Novex pre-cast polyacrylamidegel (Invitrogen) in the presence of 0.1% gelatin. Culture media (20 μl)was loaded and SDS-PAGE was performed with tris-glysine SDS buffer.After electrophoresis, the gels were washed with 5% Triton X-100 for 30minutes and stained. Protein standards were run concurrently andapproximate molecular weights were determined.

Example 1

MDA-MB 231 (ATCC) breast cancer cells were seeded on Matrigel insert inan improved Matrigel Invasion Chamber (BD). Conditioned media fromnormal human dermal fibroblast (Clonetics) supplemented with variousagents (as indicated on FIG. 1) were added to the well. The chamber wasincubated for 24 hours and the cells that had invaded the Matrigelmembrane and had migrated into the lower surface of the membrane werecounted. These data show the inhibitory effect of epigallocatechingallate and the combination of ascorbic acid, proline and lysine onMatrigel invasion and migration by MDA-MB 231 human breast cancer cells.These data also show that the combined effects of ascorbic acid, prolineand lysine in enhancing the anti-cancer activity of epigallocatechingallate.

Example 2

HTCT116 (ATCC) human colon cancer cells were seeded on Matrigel insertin an improved Matrigel Invasion Chamber (BD). Conditioned media fromNormal Human Dermal Fibroblast (Clonetics) supplemented with variousagents (as indicated on FIG. 2) were added to the well. The chamber wasincubated for 24 hours and the cells that had invaded the Matrigelmembrane and had migrated into the lower surface of the membrane werecounted. These data show an inhibitory effect of epigallocatechingallate and the combination of ascorbic acid, proline and lysine onMatrigel invasion and migration by HCT116 human colon cancer cells.These data also show that the combined effects of ascorbic acid, prolineand lysine in enhancing the anti-cancer activity of epigallocatechingallate.

Example 3

A2058 (ATCC) human melanoma cells were seeded on Matrigel insert inimproved Matrigel Invasion Chamber (BD). Conditioned media from NormalHuman Dermal Fibroblast (Clonetics) supplemented with various agents wasadded to the well. The chamber was incubated for 24 hours and the cellsthat had invaded the Matrigel membrane and had migrated into the lowersurface of the membrane were counted. These data show an inhibitoryeffect of epigallocatechin gallate and the combination of ascorbic acid,proline and lysine on Matrigel invasion and migration by A2058 humanmelanoma cells. These data also show that the combined effects ofascorbic acid, proline and lysine in enhancing the anti-cancer activityof epigallocatechin gallate.

Example 4

In this experiment A2058 (ATCC) human melanoma cells were seeded onMatrigel insert in an improved Matrigel Invasion Chamber (BD) in thepresence of:

A: conditioned media from Normal Human Dermal Fibroblast (Clonetics) and

B: the same media supplemented with various nutrients as indicated inthe FIG. 4 legends. After 24 hours of incubation the cells that invadedthe Matrigel membrane and migrated to the lower surface of the membranewere examined under the microscope and counted.

These data show an apoptotic effect of epigallocatechin gallate and thecombination of ascorbic acid+proline+lysine on A2058. A; Human MelanomaCells in tissue culture medium. B: Human Melanoma Cells in tissueculture medium containing ascorbic acid (100 μM), proline (140 μM),lysine (400 μM) and EGCG (20 μg/ml). Note: the cells were destroyed.These data also show that the combined effects of ascorbic acid, prolineand lysine in enhancing the anti-cancer activity of epigallocatechingallate.

Example 5

Cancer Cells Proliferation Study

Melanoma A2058 Cells

FIG. 5 shows the effect of 10, 20 and 50 μg/ml of EGCG without and withlysine, proline, and ascorbic acid supplementation on the proliferationof melanoma cells. Neither lysine, proline, and ascorbic acid nor EGCGat 10 and 20 μg/ml had any significant effect on cell proliferation.However, EGCG at 50 μg/ml significantly reduced the cell number to 30%.A similar effect was observed with lysine, proline and ascorbic acid.

Breast Cancer MDA-MB-231 Cells

In these experiments, the basal media was supplemented with 0, 10, 20,50, 100 or 200 μg/ml of EGCG (FIG. 6). The results show thatsupplementation of the basal media with 50, 100 and 200 μg/ml of EGCGsignificantly reduced the cell number to 66.1 ±5.3, 33.6±2 and 29.6±0.8%compared to unsupplemented controls respectively. EGCG concentrations inthe cellular media up to 20 μg/ml did not have any significantinhibitory effect on cell proliferation.

We also studied the effects of ascorbic acid, lysine, proline anddifferent concentrations of EGCG on proliferation of cancer cells. FIG.7 shows non-significantly reduced the cell number to 86.1+1.93% withascorbic acid, lysine and proline. Further addition of 20, 50 and 100 μgEGCG to this combination significantly reduced the cell number to74±5.8, 64.8±1.6 and 22±5% compared to the control group respectively.

Colon Cancer HCT116 Cells

While the inhibitory effect of ascorbic acid, proline and lysine on cellproliferation of colon cancer cells was not pronounced, the combinationof ascorbic acid, proline and lysine with 20 μg/ml EGCG significantlydecreased the cell number to 69±0.5% (FIG. 8). A higher level of EGCG inthis combination (50 μg/ml) drastically reduced the cell number to4.6±0.3%. These data also show that the combined effects of ascorbicacid, proline and lysine in enhancing the anti-cancer activity of EGCG.

The anti-proliferative activity of the nutrient combinations used inthese studies varied with the type of cancer cells. In breast cancercells, the combination of ascorbic acid, proline and lysine with EGCGhad higher anti-proliferative effects than when these nutrients wereused individually. In melanoma and colon cancer cells exposed to thecombination of ascorbic acid, proline, lysine did not affect theirproliferation. However, combining these nutrients with 20 μg/ml EGCGresulted in a significant reduction in the number of colon cancer cellsbut not in melanoma cells. The colon cancer cells appeared to be moresensitive than the breast cancer cells and melanoma cells the least tothe combination of ascorbic acid, proline, lysine and EGCG. Theproliferation of the colon cancer cells was almost completely reduced(4.6%) when ascorbic acid, proline and lysine was given with 50 μg/ml ofEGCG.

Example 6

Geltinase Zymograph Studies

The effect of EGCG on melanoma cells with basal and ascorbic acid,proline, and lysine supplementation on expression of MMPs is depicted inFIG. 9 by gelatinase zymography. Melanoma cells showed two bandscorresponding to MMP-2 and MMP-9. Ascorbic acid, lysine and prolinecombination has no effect on the expression of MMPs bands as compared tothe basal. However, EGCG inhibits the expression of both MMP-2 and MMP-9in a dose dependent manner. The intensity of bands for basal andascorbic acid, lysine and proline combination were the same. These dataalso show that the combined effects of ascorbic acid, proline and lysinein enhancing the anti-cancer activity (i.e., blocking the expression ofMMPs) of EGCG.

Example 7

Extracellular Matrix Invasion and Migration Studies

The inhibitory effects of a combination of ascorbic acid, proline andlysine used separately and together with various concentrations of EGCGwere examined. The effects of these combinations on extracellular matrixusing pre-formed Matrigel matrices, routinely used to assess invasivepotential of various cancer cell lines were studied.

Breast Cancer MDA-MB-231 Cells

FIG. 10 shows the results of Matrigel invasion of breast cancer cellsincubated in the presence of ascorbic acid, proline and lysine. Invasionof cancer cells incubated in a combination of ascorbic acid, proline andlysine was reduced to 48.1±22.1% compared to cells incubated innon-supplemented media. In the media supplemented with 20 μg/ml of EGCGonly, the number of invading cells decreased to 69.5±27.4%. The completeinhibition of matrix invasion by breast cancer cells was achieved in thepresence of higher EGCG concentrations (50 μg/ml and 100 μg/ml). Thesedata also show that the combined effects of ascorbic acid, proline andlysine in enhancing the anti-cancer activity of epigallocatechingallate.

In another series of studies, supplementation of media with 100 μMascorbic acid reduced the invasion by 36%. Supplementation with ascorbicacid and 140 μM proline further reduced the invasion by 47%. Using 400μM lysine in addition to ascorbic acid and proline in the supplementfurther reduced the invasion to 67%; lysine shows a linear response inenhancing the effects of ascorbic acid and proline up to 800 μM level.

FIG. 10 also shows that the combination of ascorbic acid, proline andlysine as well as 20 μg/ml of EGCG was effective in a complete stoppingof the invasion of cancer cells through the extracellular matrix. Thiscombination made it possible to achieve the maximum inhibitory effect oncancer cells invasion without the necessity of using high concentrationsof individual nutrients. As such, ascorbic acid, proline and lysinealong with EGCG made it possible to stop matrix invasion of breastcancer cells completely at lower level of EGCG (20 μg/ml).

Colon Cancer HCT116 Cells

FIG. 11 shows that the combination of ascorbic acid, proline and lysinesignificantly reduced the invasion of colon cancer cells to 67.2±3.7%.The EGCG used alone at 20 μg/ml reduced the invasion to 44.9±3.3%, whilethe combination of ascorbic acid, proline and lysine and 20 μg/ml ofEGCG had a synergistic effect reducing colon cancer cells invasion to24.9±4.6%.

Melanoma A2058 Cells

FIG. 12 shows that the combination of ascorbic acid, proline, and lysinewas effective in reducing the number of invading cells to 88.2±4%,however this decrease was not statistically significant. Combining thesenutrients with as little as 20 μg/ml of EGCG was effective in reducingthe number of invading cells to zero.

The results show that the use of ascorbic acid, proline and lysine withEGCG enabled us to obtain drastic reduction in the number of cellsinvading and migrating through Matrigel membrane at lower levels ofEGCG. The invasion was reduced to zero using as low a level as 20 μg/mlEGCG with ascorbic acid, proline and lysine in breast cancer cells andmelanoma cells. The benefits of the combination results were not asspectacular with colon cancer cells as obtained with breast cancercells. The level of EGCG had to be at 50 μg/ml to obtain 90% reductionin invasion by these cells. In this study, no alteration in MMPsexpression in melanoma cells was observed, although EGCG has aninhibitory effect on their expression in a dose dependent fashion.

These series of studies conclusively demonstrate that the presentnutrient pharmaceutical formulation comprising ascorbic acid, proline,lysine and at least one polyphenol compound exerts an effectiveanti-proliferative and anti-invasive effects on cancer cells.

Example 8

The following nutrient pharmaceutical formulations (Tables 1-5) andEPICAN FORTE™ (Table 6) are illustrated for their preparations. Theseformulations comprising ascorbic acid, proline, lysine and at least onepolyphenol compound are found to be effective in blocking invasion ofcancer cells and metastasis of cancer cells. TABLE 1 Formulation 1Biochemical Substances Amount % Ascorbic Acid 250 mg 5.6 CalciumAscorbate 250 mg 5.6 Magnesium Ascorbate 250 mg 5.6 Ascorbyl Palmitate250 mg 5.6 Polyphenols 1,000 mg 22.5 N-Acetyl-Cysteine 200 mg 4.5 Lysine1,000 mg 22.5 Proline 750 mg 16.8 Arginine 500 mg 11.2 Selenium 30 mcg<0.01 Copper 2 mg 0.04 Manganese 1 mg 0.02mg = milligramsmcg = micrograms“%” refers to % weight of individual ingredient over total weight offormulation

TABLE 2 Formulation 2 Biochemical Substances Amount % Ascorbic Acid 25mg 5.7 Calcium Ascorbate 25 mg 5.7 Magnesium Ascorbate 25 mg 5.7Ascorbyl Palmitate 25 mg 5.7 Polyphenols - 50% 200 mg 46.0N-Acetyl-Cysteine 10 mg 2.3 Lysine 50 mg 11.5 Proline 25 mg 5.7 Arginine50 mg 11.5 Selenium 1 mcg <0.001 Copper 20 mcg <0.001 Manganese 50 mcg<0.001mg = milligramsmcg = micrograms“%” refers to % weight of individual ingredient over total weight offormulation

TABLE 3 Formulation 3 Biochemical Substances Amount % Ascorbic Acid5,000 mg 13.3 Calcium Ascorbate 5,000 mg 13.3 Magnesium Ascorbate 5,000mg 13.3 Ascorbyl Palmitate 5,000 mg 13.3 Polyphenols - 100% 5,000 mg13.3 N-Acetyl-Cysteine 1,500 mg 4.0 Lysine 5,000 mg 13.3 Proline 3,000mg 8.0 Arginine 3,000 mg 8.0 Selenium 200 mcg <0.001 Copper 9 mg 0.02Manganese 10 mg 0.002mg = milligramsmcg = micrograms“%” refers to % weight of individual ingredient over total weight offormulation

TABLE 4 Formulation 4 Biochemical Substances Amount % Ascorbic Acid 250mg 4.7 Calcium Ascorbate 250 mg 4.7 Magnesium Ascorbate 250 mg 4.7Ascorbyl Palmitate 250 mg 4.7 Polyphenols - 98% 1,000 mg 18.7N-Acetyl-Cysteine 200 mg 3.7 Lysine 1,000 mg 18.7 Proline 750 mg 14.0Arginine 500 mg 9.3 Selenium 100 mcg <0.002 Copper 2 mg 0.04 Manganese 1mg 0.02 Calcium 500 mg 9.3 Magnesium 400 mg 7.5mg = milligramsmcg = micrograms“%” refers to % weight of individual ingredient over total weight offormulation

TABLE 5 Formulation 5 Biochemical Substances Amount % Ascorbic Acid 250mg 4.5 Calcium Ascorbate 250 mg 4.5 Magnesium Ascorbate 250 mg 4.5Ascorbyl Palmitate 250 mg 4.5 Polyphenols - 98% 1,000 mg 18N-Acetyl-Cysteine 200 mg 3.6 Lysine 1,000 mg 18 Proline 750 mg 13.5Arginine 500 mg 9 Selenium 100 mcg <0.002 Copper 2 mg <0.04 Manganese 1mg <0.02 Calcium 500 mg 9 Magnesium 400 mg 7.2 Citrus Bioflavonoids 200mg 3.6mg = milligramsmcg = micrograms“%” refers to % weight of individual ingredient over total weight offormulation.

TABLE 6 Formulation for EPICAN FORTE ™ Serving size: 6 Capsules(Suggested use: two capsules three times daily preferably with meals)Amount per 6 Capsules contain: serving % Daily Value L-Lysine 1,000 mgL-Proline 750 mg L-Arginine 500 mg Vitamin C (as Ascorbic Acid, Calcium782 mg 1180% Ascorbate, Magnesium Ascorbate, and Ascorbate Palmitate)Calcium (from Calcium Ascorbate) 22 mg   2% Magnesium (from MagnesiumAscorbate) 50 mg  12% Standard Green Tea Extract (leaf) 1,000 mg (80%polyphenol's - 800 mg) decaffeinated N-Acetyl-Cysteine 200 mg Selenium(from L-Selenomethionine) 30 mg  43% Copper (from Copper Glycinate) 2 mg 100% Manganese (from Manganese Citrate) 1 mg  50%mg = milligramsmcg = microgramsEPICAN FORTE ™ is a trademark name under Matthias Rath, Inc. in apending U.S. application.Other ingredients:Vetetarian capsules (hydroxypropyl methycellulose), silicon dioxide,cellulose and magnesium stearate.

Example 9 Effects of EPICAN FORTE™ on Human Cancer Cells

The effects EPICAN FORTE™ on human cancer cells were studied. Metastaticparameters such as expression of matrix metalloproteinases (MMPs) bygelatinase zymography, invasion potential through Matrigel andproliferation/growth by MTT assays were studied. The protocols for theseassays are described in detail as above. Several human cancer cell lineswere used: skin cancer—melanoma cells 2058, liver cancer—HepG2 cells,fibrosarcoma—HT 1080 cells, colon cancer—HCT 116, breast cancerER+/−MCF-7 and breast cancer ER−/−MDA-MB-231.

The following tables (Tables 7 and 8) summarize the results:

Effects of EPICAN FORTE™ on Proliferation/Growth of Human Cancer CellLines: TABLE 7 Treatment Doses of EPICAN FORTE ™ (μg/ml): Percent ofControl Cancer Cells 0 10 50 100 500 1000 Melanoma 100% 98 — 105 — 50Hep G2 100% 92 — 138 — 85 HT-1080 100% 100 — 97  80 63 Colon 100% 119125  141 120 106 (HCT 116) MCF-7 100% 93 88 92  97 77 MDA-MB-231 100%103 — 82 — 25

Effects of EPICAN FORTE™ on Matrigel Invasion and Migration by HumanCancer Cells: TABLE 8 Treatment Doses of EPICAN FORTE ™ (μg/ml): PercentInhibition Cancer Cell 0 10 50 100 500 1000  Melanoma 0% 80 98 100 — —HepG2 0% 15 25 50  95 100 HT-1080 0% 90 — 50  70 100 Colon 0% 20 50 80100 — (HCT 116) MDA-MB-231 0% 50 — 98 — — MCF-7 Non-invasive

Effects of EPICAN FORTE™ on the Expression of Matrix Metalloproteinases(MMPs) by Human Cancer Cell Lines:

Melanoma Cells: Melanoma cells exhibited two bands on gelatinasezymography corresponding to MMP-2 and MMP-9. EPICAN FORTE™ inhibited theexpression of MMP-2 and -9 in a dose dependent fashion. The expressionof MMP-2 and -9 was significantly inhibited with a concentration of 100μg/ml of EPICAN FORTE™ and virtually undetectable with a concentrationof 1000 μg/ml.

HepG2 Cells: Like melanoma cell, HepG2 cell also expressed two bandscorresponding to MMP-2 and MMP-9. EPICAN FORTE™ inhibited the expressionof MMP-2 and-9 at 500 and 1000 μg/ml concentration.

Fibrosarcoma HT-1080: HT-1080 cells exhibited two bands for MMP-2 and-9. EPICAN FORTE™ also inhibited the expression of both the bands in adose dependent fashion. Very faint bands were seen at 500 and 1000 μg/mlconcentration.

Colon cancer HCT 116: Colon cancer cells showed only one band onzymography corresponding to MMP-2 which totally disappeared at 100 μg/mlconcentration.

MCF-7 and MDA-MB-231: These cancer cell lines did not any MMPs bandsunder our experimental concentration, which were similar to the othercancer cell lines.

Invasion of MDA-MB-231 through Matrigel was inhibited by 50, 60 and 95%by 10, 50 and 100 μg/ml of EPICAN FORTE™ respectively. EPICAN FORTE™ wasnot toxic to MDA-MB-231 at 10 μg/ml, showed slight toxicity at 100μg/ml. However, it exhibited significant toxicity at 1000 μg/ml. NeitherMMP-2 nor MMP-9 was expressed by zymography. In contrast, EPICAN FORTE™was not toxic to MCF-7 even at 500 μg/ml, and exhibited slight toxicityat 1000 μg/ml. MCF-7 was not invasive and did not express MMPsactivities.

EPICAN FORTE™ inhibits the expression of MMP-2 and MMP-9 in adose-dependent fashion. The expression of MMP-2 and MMP-9 wassignificantly inhibited with a concentration of 100 μg/ml of EPICANFORTE™ and virtually undetectable with a concentration of 100 μg/ml.EPICAN FORTE™ used at 10 and 100 μg/ml concentrations did notsignificantly affect the cells viability, and at 100 μg/ml it showedcytotoxicity at the range of 10-40 percent, depending on cell type. Theinvasion of melanoma cells, MDA-MB-231 cells, and a co-culture ofmelanoma cells with NHDF through Matrigel was significantly reduced in adose-dependent manner. Invasion of HT-1080 cells thought Matrigel wasinhibited by 10%, 50%, 70% and 100% at 10, 100, 200 and 1000 μg/mlrespectively. Interestingly, EPICAN FORTE™ was not toxic to HT-1080cells at 100 μg/ml. These results demonstrate that EPICAN FORTE™ is veryeffective for several cancer cell lines and also in co-culture. Theseobservations reveal that EPICAN FORTE™ may provide a natural therapeuticbasis that makes it a valuable and promising candidate for the treatmentof human cancers.

Example 10 Effects of EPICAN FORTE™ on Human Normal Cell Lines

The effects of EPICAN FORTE™ on normal human cells was studied.Parameters similar to those of cancer cell lines were employed; namelyzymography for MMPs expression, invasion through Matrigel andproliferation/growth by MTT assays. Several normal human dermalfibroblast (NHDF) were used, including: human chondrocytes and humanstromal cells.

The following tables (Tables 9 and 10) summarize the effects of EPICANFORTE™ on proliferation/growth and metastasis invasion in normal humancells: TABLE 9 Effects of EPICAN FORTE ™ on Proliferation/Growth ofNormal Human Cells. Treatment Doses of EPICAN FORTE ™ (μg/ml): Percentof Control Normal Cells 0 10  50 100 500  100 NHDF 100% 118 — 135 — 90Chondrocytes 100% 113 — 148 76 82 Stromal Cells 100% 100 116 115 98 64

TABLE 10 Effects of EPICAN FORTE ™ on Matrigel Invasion by Normal HumanCells. Treatment Doses of EPICAN FORTE ™ (μg/ml): Percent InhibitionNormal Cell 0 10 50 100 200 1000 NHDF 0% 67% 89% 100%  — — Chondrocytes0% 45% 85% 95% 100% — Stromal Cells 0%  0% — 95% 100%

The effects of EPICAN FORTE™ on the expression of matrixmetalloproteinases (MMPs) by normal human cells are summarized asfollowed:

NHDF: NHDF expressed only one band on zymography corresponding to MMP-2,which virtually disappeared at 1000 μg/ml concentration of EPICANFORTE™.

Chondrocytes: Chondrocytes also showed only one band corresponding toMMP-2. EPICAN FORTE™ inhibited the expression of MMP-2 in a dosedependent manner. The expression of MMP-2 was significantly inhibited ata concentration of 100 μg/ml of EPICAN FORTE™ and totally disappeared at200 μg/ml concentration of EPICAN FORTE™.

Stromal Cells: Stromal cells also exhibited only one band correspondingto MMP-2. EPICAN FORTE™ inhibited the expression of MMP-2 in a dosedependent fashion. Very faint band was seen at 50 and 100 μg/mlconcentration which was undetected at 200 and 500 μg/ml concentration.

In summary, the results indicated that EPICAN FORTE™ inhibits theexpression of MMP-2 in a dose-dependent manner. The expression of MMP-2was significantly inhibited at a concentration of 100 μg/ml of EPICANFORTE™ and virtually not detected at a concentration of 200 μg/ml. Inaddition, it was also found that invasion of chondrocytes throughMatrigel was inhibited by 50%, 85% and 95% at 10 μg/ml, 100 μg/ml and200 μg/ml. At 500 μg/ml the invasion was totally reduced to 0%.

EPICAN FORTE™ was not toxic to chrondrocytes even at a concentration of200 μg/ml. In fact, EPICAN FORTE™ exerted a cell proliferation effect, a70% increase in cell proliferation with 200 μg/ml, 70% more over thecontrol. Slight toxic effect was seen only at 500 μg/ml. These resultsdemonstrate that EPICAN FORTE™ is effective in inhibiting the MMP-2expression and that EPICAN FORTE™ represents a novel anti-inflammatorynutrient formulation as a natural approach to inhibit MMP production andextracellular matrix degradation in osteoarthritis and other relateddisorders including excessive cartilage degradation.

Example 11

Female patient was diagnosed with Gliablastoma after suffering a facialstroke characterized by numbness of lips and the left side of the face.MR scans (FIG. 13; left diagram) revealed a brain tumor on the left sideof the brain as shown in the left scan. Patient was treated withradiation treatment. Side effect included falling out of the hair.Patient also took cortisone, which resulted in water retention andbloating and an eventual weight gain of approximately 20 kg. Follow upMR scans revealed that despite the treatments, there was no reduction inthe size of the tumor. Prognosis by treating physicians was eventualdeath as a result of continued tumor growth.

On Apr. 10, 2002, patient started oral administration of the EPICANFORTE™ formula. Dosage was between 6-9 capsules per day. Patient thendiscontinued taking cortisone and lost over 20 kg. Patient continued theEPICAN FORTE™ treatment for approximately 5 months. MR scan (FIG. 13;right diagram) of the patient brain in August of 2002 could no longerdetect the brain tumor.

Example 12

Male patient was found to have a markedly elevated level (59.9 μg/l) ofthe tumor marker PSA and was diagnosed with prostate cancer. The X-rayCT scan revealed metastasis along the lymphatic vessels of the aorta(FIG. 14; left diagram).

After diagnosis started oral administration of the EPICAN FORTE™formula. Dosage was between 6-9 capsules per day. After approximatelythree months of treatment with EPICAN FORTE™, the patient's PSA levelfell from 59.9 μg/l to 0.9 μg/l.

On Oct. 23, 2002, approximately 5 months from initiating administrationof the EPICAN FORTE™ formula, the patient was given a follow up CT scan.The previously visible lymphatic metastasis was no longer detectable(right diagram), and the patient's prostate returned to normal size.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention. w

1-34. (canceled)
 35. A nutrient pharmaceutical composition useful intreating cancer, comprising: a) an ascorbic compound; b) an amino acidcombination consisting essentially of L-Lysine, L-proline, and N-acetylcysteine; and c) at least one polyphenol compound selected from thegroup consisting of epigallocatechin gallate, epicatechin gallate,epigallocatechin, epicatechin, and catechin, wherein the compounds of a)and b) enhance the polyphenol compound's activity in blocking cancerproliferation and metastasis.
 36. The nutrient pharmaceuticalcomposition of claim 35, wherein the ascorbic compound is selected fromthe group consisting of ascorbic acid, pharmaceutically acceptableascorbate salts, ascorbate esters and/or mixture thereof.
 37. Thenutrient composition of claim 36, wherein the pharmaceuticallyacceptable ascorbate salt is calcium ascorbate or magnesium ascorbate.38. The nutrient composition of claim 36, wherein the ascorbate ester isascorbyl palmitate.
 39. The nutrient pharmaceutical composition of claim35, wherein the lysine compound is selected from the group consisting oflysine hydrochloride and pharmaceutically acceptable lysine salts. 40.The nutrient pharmaceutical composition of claim 35, wherein the prolinecompound is selected from the group consisting of proline hydrochlorideand pharmaceutically acceptable proline salts.
 41. The nutrientpharmaceutical composition of claim 35, further comprising a traceelement selected from the group consisting of selenium, copper,manganese, calcium and magnesium.
 42. The nutrient pharmaceuticalcomposition of claim 35, further comprising an amino acid.
 43. Thenutrient pharmaceutical composition of claim 42, wherein the amino acidis arginine.
 44. The nutrient pharmaceutical composition of claim 35,further comprising N-acetyl cysteine.
 45. A nutrient pharmaceuticalcomposition useful in treating cancer, comprising: ascorbic acid,calcium ascorbate, magnesium ascorbate, ascorbyl palmitate, polyphenols,N-acetyl-cysteine, lysine, proline, arginine, selenium, copper, andmanganese.
 46. The nutrient pharmaceutical composition of claim 45,wherein the composition comprises 250 mg ascorbic acid, 250 mg calciumascorbate, 250 mg magnesium ascorbate, 250 mg ascorbyl palmitate, 1,000mg polyphenols, 200 mg N-acetyl-cysteine, 1,000 mg lysine, 750 mgproline, 500 mg arginine, 30 mcg selenium, 2 mg copper, and 1 mgmanganese.
 47. The nutrient pharmaceutical composition of claim 45,wherein the composition comprises 25 mg ascorbic acid, 25 mg calciumascorbate, 25 mg magnesium ascorbate, 25 mg ascorbyl palmitate, 200 mgpolyphenols, 10 mg N-acetyl-cysteine, 50 mg lysine, 25 mg proline, 50 mgarginine, 1 mcg selenium, 20 mcg copper, and 50 mcg manganese.
 48. Thenutrient pharmaceutical composition of claim 45, wherein the compositioncomprises 5,000 mg ascorbic acid, 5,000 mg calcium ascorbate, 5,000 mgmagnesium ascorbate, 5,000 mg ascorbyl palmitate, 5,000 mg polyphenols,1,500 mg N-acetyl-cysteine, 5,000 mg lysine, 3,000 mg proline, 3,000 mgarginine, 200 mcg selenium, 9 mg copper, and 10 mg manganese.
 49. Thenutrient pharmaceutical composition of claim 45, wherein the compositioncomprises 250 mg ascorbic acid, 250 mg calcium ascorbate, 250 mgmagnesium ascorbate, 250 mg ascorbyl palmitate, 1,000 mg polyphenols,200 mg N-acetyl-cysteine, 1,000 mg lysine, 750 mg proline, 500 mgarginine, 100 mcg selenium, 2 mg copper, 1 mg manganese, 500 mg calcium,and 400 mg magnesium.
 50. The nutrient pharmaceutical composition ofclaim 45, wherein the composition comprises 250 mg ascorbic acid, 250 mgcalcium ascorbate, 250 mg magnesium ascorbate, 250 mg ascorbylpalmitate, 1,000 mg polyphenols, 200 mg N-acetyl-cysteine, 1,000 mglysine, 750 mg proline, 500 mg arginine, 100 mcg selenium, 2 mg copper,1 mg manganese, 500 mg calcium, 400 magnesium, and 200 citrusbiofavonoids.
 51. A nutrient pharmaceutical composition useful intreating cancer, comprising: L-lysine, L-proline, L-arginine, ascorbicacid, calcium, magnesium, polyphenols, N-acetyl-cysteine, selenium,copper, and manganese.
 52. The nutrient pharmaceutical composition ofclaim 51, wherein the composition comprises 1,000 mg L-lysine, 750 mgL-proline, 500 mg L-arginine, 710 mg ascorbic acid, 22 mg calcium, 50 mgmagnesium, 1000 mg polyphenols, 200 mg N-acetyl cysteine, 30 mcgselenium, 2 mg copper, and 1 mg manganese.
 53. The nutrientpharmaceutical composition of claim 52, wherein the ascorbic acid isselected from the group consisting of ascorbic acid, calcium ascorbate,magnesium ascorbate and ascorbyl palmitate.
 54. The nutrientpharmaceutical composition of claim 52, wherein the polyphenol isselected from the group consisting of epigallocatechin gallate,epicatechin gallate, epigallocatechin, epicatechin, catechin and otherpharmaceutically acceptable polyphenol salts and/or mixtures thereof.